Insulin-stimulated glucose transport activity in rat adipocytes is inhibited by isoproterenol and enhanced by adenosine. Both of these effects occur without corresponding changes in the subcellular distribution of the GLUT4 glucose transporter isoform. In this report, we have utilized the impermeant, exofacial bis-mannose glucose transporter- specific photolabel, 2-N-4- (1-azi-2,2,2-trifluoroethyl)benzoyl-1, 3-bis (D-mannos-4-yloxy) -2-propylamine (ATB-BMPA), to examine the cell surface accessibility of GLUT4 glucose transporters under these conditions. compared to cells treated with insulin alone, adenosine in the presence of insulin increases the accessibility of GLUT4 to the extracellular photolabel by approximately 25% consistent with its enhancement of insulin-stimulated glucose transport activity; the plasma membrane concentration of GLUT4 as assessed by Western blotting is unchanged. Conversely, isoproterenol, in the absence of adenosine, promotes a time- dependent (t[1/2] equals approximately 2 min) decrease in the accessibility of insulin-stimulated cell surface GLUT4 by >50%, which directly correlates with the observed inhibition of transport activity; the plasma membrane concentration of GLUT4 decreases by 0-15%. Photolabeling the corresponding plasma membranes revealed that these alterations in the ability of the photolabel to bind to GLUT4 are transient as the levels of both photolabel incorporation and plasma membrane glucose transport activity were consistent with the observed GLUT4 concentration. These data suggest that insulin-stimulated GLUT4 glucose transporters can exist in two distinct states within the adipocyte plasma membrane, one functional and accessible to extracellular substrate and one non-functional and unable to bind extracellular substrate. These effects are only observed in the intact adipocyte and are not retained in isolated plasma membranes isolated from these cells when analyzed for the ability to transport glucose or bind photolabel.